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Approaches to limiting the dispersal of transgenic potatoes

(2008 – 2011) Friedrich-Alexander University Erlangen-Nürnberg, Chair of Biochemistry

Topic

The aim of this project is to develop a method to limit the dispersal of potatoes using different genetic engineering approaches (biological confinement). Potatoes propagate via tubers which germinate after a period of dormancy. This process can be suppressed by introducing suitable gene constructs. The cultivation of these transgenic potatoes could prevent tubers left on the field after harvesting (volunteers) sprouting in the following year, resulting in the undesirable spread of potatoes. However, it must be possible to interrupt this period of dormancy as and when required so that the tubers can be used as seed potatoes. This can be achieved by inserting a second gene construct which reverses the effect of the first. The activation of the second construct can be controlled externally using a molecular switch, which will allow the time of germination to be determined.

Experiment description

The dormancy of potato tubers can be influenced by the expression of a transgene. Two wild-type potato tubers sprouting approx. 8 weeks after harvesting (left). The transgenic tubers show no signs of shoot growth at the same stage.

The aim is to produce transgenic potato plants that form non-germinating tubers using various approaches, and then to analyse them. Three metabolic processes which are necessary for germination will be genetically modified at key stages.

Carbohydrate metabolism: The concentration of an important sugar (trehalose-6-phosphate) present in potatoes will be modified. This sugar influences the potato’s ability to mobilise nutrients required for germination. The greater the quantity of this sugar is available to the tuber, the longer it can remain dormant. Dormancy can be interrupted by reducing the level of trehalose-6-phosphate.

The concentration of trehalose-6-phosphate will be controlled by inserting two genes into the potato plants. One gene from the bacterium E.coli expresses an enzyme in the tuber which produces this sugar. The other gene causes an enzyme to be produced which breaks down the sugar again so that germination can start. To make sure that dormancy can be interrupted when required, the second gene is controlled by a promoter which is expressed only when alcohol (vapour) is added.

Plant hormones: The duration of dormancy is also controlled by the concentration of plant hormones. The production and breakdown of two plant hormones (gibberellins, GA and cytokinins, CK) therefore represents another possible method of producing non-germinating potato tubers. The production and breakdown of these hormones will also be controlled in two stages. Initially, the aim is to suppress germination through the expression of enzymes that break down hormones. Some of these enzymes will be produced in the tubers, whilst others will be produced only in the tubers’ meristem tissue. To trigger germination, the enzyme-producing genes will be switched off again in a second step (gene silencing).

Potato plants will be transformed with the corresponding gene constructs and then analysed using molecular-biological techniques (PCR and Northern Blot). The strains which have absorbed the constructs will be propagated in the greenhouse and where they will remain until the tubers have formed. The harvested tubers will be observed over an extended period of time to determine the potential effects on the length of dormancy. Strains producing non-germinating tubers will then be transformed with the second gene construct and will once again remain in the greenhouse until the tubers form. These genes will also be controlled by a promoter which is expressed only when alcohol (vapour) is added.

Meristem development: When tubers germinate, the tissue in the tubers changes in the region where the shoot subsequently forms (meristem). A large number of genes are involved in this process, two of which have been identified in preliminary research and selected for further investigation. The silencing of these genes could suppress the germination of the tubers. Potato plants will be genetically modified to achieve this effect and then analysed using a molecular-biological technique (PCR). The transgenic plants in which the selected genes have been strongly suppressed will be grown on in the greenhouse. The germination behaviour of the tubers from these plants will then be observed for an extended period of time.

The successful methods will then be field tested. This will involve placing the transgenic tubers on the field in the autumn and comparing their germination behaviour with that of wild-type tubers the following year. Additional studies will be conducted to analyse the germination behaviour of the transgenic tubers during storage.

Results

Carbohydrate metabolism: Transgenic potato tubers were produced in which the concentration of trehalose-6-phosphate (T6P) was modified by the introduction of the new genes. These tubers were then compared with the wild type in terms of their seed dormancy. It was demonstrated that a targeted modification of the T6P concentration influenced the seed dormancy of the potato tubers.

Biochemical analyses showed that the starch content of the tubers with the sugar-producing enzyme was 20 – 30 per cent lower. The starch content of the potato tubers with the sugar-breakdown enzyme was unchanged.

At a later stage of the project, potato tubers were produced that contained both gene constructs. This double transformation and subsequent application of alcohol vapour breaks the delayed seed dormancy. A PCR test was used to confirm the presence of the two gene constructs. These tubers are currently being matured in the greenhouse so they can be harvested and examined.

Plant hormones: Transgenic potato plants were created that produce more hormone-degrading enzymes. These enzymes, which influence the breakdown of gibberellin and cytokinin plant hormones, were produced in all parts of the plant under the control of a permanently active promoter. The tubers displayed a longer seed dormancy – some of them slightly longer, others much longer – but the parts of the plant above ground showed morphological changes. To avoid this, promoters were used that ensure that the enzymes are produced only in the tubers, or in the meristem. After regeneration in the greenhouse, these plants exhibited no morphological changes to their above-ground parts.

Several lines were identified that produce more of the hormone-breakdown enzyme only in their tubers. The impact of the transgenes on seed dormancy is currently being investigated. In transgenic plants that produce one of the two enzymes (gibberellins) in the meristem, expression was detected using PCR. Following regeneration, the outward appearance of the plants and tubers was not noticeably different.

Meristem development: Potato plants were genetically modified in such a way that two of the genes involved in the meristem development process were silenced. However, attempts to regenerate transgenic plants from this transformation were only partially successful because silencing one of the two genes caused the plants to die. Research using this gene was therefore discontinued. 32 transgenic potato plants were regenerated from the transformation with the second gene construct. These are currently undergoing molecular biological tests.