Aug 5, 2005

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Testing and further development of microinjection as a new transformation method in plants

(2001 – 2004) University of Giessen, Institute of Botany

Topic

The aims of this project were:

  • to further develop microinjection as a method of transformation so that it can be used for plant cells as well;
  • and thereby to establish a procedure with which it is possible to transfer only the functionally required genes without using marker genes.

In collaboration with other participants in the joint project, this method was to be applied and tested for various reporter genes and for two genes of economic interest.

Information on the procedure:

Summary

It proved possible to produce transgenic calli. However, without a selective marker, it has not yet been possible to generate transgenic plants.

When the DNA really penetrated the cell nucleus, this resulted in a very high transformation rate. In practice this is not always the case, however, but it can be checked in a simple way.

Experiment description

Using fine canulae, DNA that codes for a fluorescent protein was inserted into onion cells. Two days after the injection the green dye can be detected. Integration of the foreign DNA into the onion genome was successful.Microinjection is a procedure for i

Using fine cannulae, DNA which codes for a fluorescent protein was injected into onion cells. Two days after the injection the green dye is visible. Integration of the foreign DNA into the onion genome was successful.

Microinjection was used to insert DNA into onion cells. The gene product can be detected by means of the blue colouring. Only the cell in the centre was injected. The gene product also made its way to the surrounding cells through small connecting channel

DNA was inserted into onion cells by means of microinjection. The gene product is verifiable via the blue colour. Only the cell in the centre was injected. The gene product also travelled to the surrounding cells through small connecting channels.

Photos: Michael Knoblauch, University of Giessen

Oilseed rape and potatoes were used as model plants for testing microinjection. Various genes were injected into their cells. To this end, the procedure was adapted to the special features of plant cells during earlier experiments. These involved e.g. using a specially formed cannula, developing a pressure generator to prevent loss of pressure in the cell after inserting the cannula, and controlling the release of the injected substance.

The purpose of the current project was to further optimize and test microinjection as a transformation method for plant cells. The process included the following stages:

Development of parameters which influence transformation success, such as: injected DNA quantity, the pressure necessary for this, and choice of cell components which are particularly suited to injection.

Various approaches were tested in order to increase the reliability of the procedure and the transformation rate:

  • Agroinjection experiment:Microinjection in the presence of certain proteins from Agrobacterium tumefaciens which are involved in the integration of T-DNA into the plant genome.
  • Use of special restriction enzymes which support integration of the DNA
  • Activation of the cell’s own DNA repair system (with UV-radiation) to increase the transformation efficiency

Marker genes are not necessary when using microinjection, since a fluorescent dye is injected in parallel with the DNA which stays in the vacuole inside the cell for a long time, thus acting as a marker for the transformed cell.

Results

To date, DNA has been injected into the cell nucleus and cytoplasm of potatoes. Fine, microscopy-compatible potato slices were previously cut from special plant tissue (section between two shoot axes) in which the cell nuclei were easily accessible.

Transgenic calli were produced. However, it has not yet proved possible to produce transgenic plants without a selective marker.

The experiment showed that very high transformation efficiency is achieved when the DNA actually penetrates the cell nucleus. However, this is not always the case in practice. The fluorescent dye used (Texas Red) can migrate passively into the cell nucleus after injection into the cytoplasm. This simulates injection into the nucleus. However, if the DNA to be injected is dyed, this problem can be avoided in a simple manner.

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