Field trial with genetically modified petunias

In search of a rare event

On a trial field near Rostock, white and lilac-coloured petunias are growing in carefully planted adjacent rows. The ornamental plants are being used as a model plant for an outcrossing experiment with fixed roles. The petunias with the white flowers are the mother plants. They have been conventionally bred. The role of father is played by the lilac-coloured petunias, which produce pollen and have been genetically modified, although not in the usual way. In these plants the foreign gene has been inserted in the plastids instead of in the cell nucleus.

The trial field with petunias

Patricia Horn, a PhD student in Rostock University’s Department of Agroecology, conducts the experiments.

Plastids are small units of a plant cell that have their own DNA. Photograph: Prof. Hans-Ulrich Koop, LMU Munich Foto: Prof. Hans-Ulrich Koop, LMU München

Genetically modified petunia with ripe pollen that can be used for hand pollination

Outcrossing experiments are conducted by hand. First, the scientists collect genetically modified lilac-coloured flowers with ripe pollen.

Hand pollination: The stigma of a white conventional plant is pollinated with pollen from a genetically modified petunia.

The pollen from these flowers is then used to pollinate the stigma of the white mother plants.

Harvesting ripe seed capsules

After around four weeks, seed capsules form and are harvested,…

Harvested seed capsules

…assessed according to size and cleaned in the laboratory.

GUS test: If the new genetic information contained in the plastids has been passed on via the pollen, the seedling turns blue.

Seedlings are grown from the seeds and subjected to GUS staining. If a blue dye forms, it indicates a transfer of the plastid DNA via pollen.

The seedlings grown from the seeds are assessed individually.

After GUS staining: The seedlings are inspected for any blue colouration.

The project is part of the CONFICO joint project on “Developing and testing plastid transformation as a confinement system in oilseed rape and maize”. The coordinator is Prof. Dario Leister of Ludwig-Maximilians-Universität München. Other project partners are the Cell Biology and Cell Culture group led by Prof. Hans-Ulrich Koop at the University of Munich, the Chair of Genetics (Prof. Alfons Gierl) of Technische Universität München and the Chair of Agrobiotechnology (Prof. Inge Broer) of the University of Rostock.

Plastids are small units of a plant cell that have their own DNA. The genetic information in the plastids is not passed on via pollen, which means it can be confined within the plant (biological confinement). However, since we know from other research work that the spread of plastid DNA via pollen cannot be ruled out entirely, scientists at the University of Rostock are now investigating under field conditions how frequently, if at all, plastid genes could be transferred via pollen.

For the outcrossing experiments, around 8700 white and lilac-coloured petunias were planted in long rows next to each other in August last year. “In the middle of October there was the threat of severe frost at night, so we had to move all the plants to the greenhouse immediately. Otherwise the still unripe seeds would have been destroyed,” explains Patricia Horn, a PhD student at the University of Rostock. She and her two technical assistants drive to the trial field, which was planted in May this year, two or three times a week. Ms Horn is please with the way the trial is going: “Today we are lucky. The storm that was forecast for last night didn’t materialise and there are still enough flowers for pollination to take place,” she says. “We pollinate the plants by hand, so we know precisely which plants are the parents of the new seedlings.” The plants can of course reproduce without the help of the scientists, but this often leads to self-pollination, which is not wanted in this experiment.

A time-consuming manual operation

On the field, the usual routine tasks are waiting to be carried out. Lilac-coloured flowers with pollen that is ripe enough for pollination are collected. White flowers are ‘castrated’ before they open and are pollinated with transgenic pollen from the lilac-coloured flowers. “Last year we pollinated around 12,000 petunia flowers by hand. We needed three additional assistants. This year we will be pollinating 2500 flowers. First it was too hot and not enough pollen was formed, and now it keeps raining.” Patricia Horn carefully plucks off the petals and stamen from a white flower and exposes the female part, the pistil, for pollination. Then the stigma is repeatedly dabbed with pollen from a transgenic lilac-coloured flower. The hand-pollinated plants are marked, so that they can be identified at all times. A field a bit further away from the trial field contains petunias where both the pollen donors and the pollen recipients have been conventionally bred. Here too, the flowers are being artificially pollinated as a control.

Today the researchers are also harvesting seed capsules. These form around four weeks after pollination. The seeds are used to grow seedlings in the greenhouse, which are then analysed to see whether a gene transfer of plastid DNA has occurred via the pollen. The genetically modified pollen donor plants contain marker genes in the plastid genome. One of them is a GUS reporter genes. This makes it possible to carry out a simple colour test that can detect an undesirable transfer of plastid DNA.

The researchers plan to test at least 300,000 seeds in total in order to obtain a statistically secure result about the probability of pollen transfer events involving plastid DNA.

A colour test provides an initial indication of gene transfer

After harvesting, the seed capsules are cleaned in the laboratory and assessed by size. A defined quantity of seeds is placed on a nutritive medium. They need two weeks in the dark in the greenhouse at temperatures of 18-24 ºC in order to produce seedlings.

For the colour test, they are exposed to a special solution for about an hour in a vacuum. The vacuum causes the substrate to be drawn into the plant cells. The cells containing the GUS gene are able to split the substrate and form a blue pigment.

At this point, the seedlings are still very small and any colour reaction would take place only in a few cells. This means that the researchers have to look carefully for the tiny blue areas using magnifier glasses.

If they find any, they freeze the seedling in liquid nitrogen. Later on, PCR, a molecular biological method, is used to check whether the gene sequence for the GUS gene is in fact present in the cells. “This step is vital because unfortunately, false positive colour reactions occur frequently,” reports Patricia Horn. The plant cells contain enzymes that can also cause a blue colouration.

Last year around 35,000 seeds and 7,700 seedlings were analysed. However, the GUS test and subsequent molecular biological analysis identified only a few seedlings containing transgenic GUS sequences. Whether these results stand up to further analysis will be determined in the ongoing experiments.

However, the staff still have a lot of work to do to obtain the 300,000 seeds required for the next stage of the research.