May 9, 2006

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Gene transfer methods

Microinjection: genes by injection

A procedure that is now almost routine with animal cells and in medicine has so far proved very difficult with plant cells: the direct injection of new genes using ultra-fine cannulae. But microinjection would be an elegant way of avoiding marker genes.

During microinjection, DNA is injected directly into the cell, or even into the cell nucleus via an inserted cannula. The process is observed and controlled under the microscope. The DNA is then integrated into the plant genome – probably during the cell’s own DNA repair processes.

Microinjection into the cell of a potato callus.

Using fine cannulae, DNA that codes for a fluorescent protein was inserted into onion cells. Two days after the injection, the green dye can be seen. The integration of the foreign DNA into the onion genome was successful.

Potos: Michael Knoblauch, University of Giessen

The advantage of microinjection is that with this method, genetic modification in theory no longer requires a marker gene.

• The target gene, which confers a new trait, is introduced directly into a single cell.
• The cells transformed in this way are easy to identify if a dye is injected along with the DNA (see photo).
• If the process works, it will no longer be necessary to select the transformed cells using antibiotic resistance or herbicide resistance markers.

So far this method has been used primarily with animal cells. One of the ways in which plant cells differ from animal cells is that they have a stable cell wall. To use microinjection with plants the method has to be adapted to their special characteristics.

• The most important difference is the cell wall and the internal cell pressure of up to 30 bar. If the kind of cannula used for animal cells is used on plant cells it causes a drastic loss of pressure and the cell dies. To solve the problem, even thinner cannula tips are being developed and used in combination with a finely adjustable pressure generator. In this way the cannula enters the plant cell without destroying it.
• Another approach to improving microinjection as a transformation method for plant cells focuses on the efficiency with which the injected genes are integrated into the plant genome. One way of improving this is to inject special proteins along with the target gene sequences which support the stable integration of ‘foreign’ genes into the plant genome and are produced by Agrobacterium tumefaciens (agroinjection). This soil bacterium is naturally capable of inserting its own genes into the genome of plants.

Results: Several biosafety research projects have looked at microinjection in plants and it has been successfully established as a new transformation method.

However, it was not possible in all cases to achieve high transformation efficiency. In addition, it was possible to regenerate transgenic plant cells into plants following microinjection only when a marker gene (kanamycin resistance) had been introduced. For the time being then, even microinjection cannot do without marker genes.

By contrast, agroinjection is not necessary in order to produce transgenic plants using microinjection. In principle, this opens up the possibility of using this method commercially at a low cost.

Microinjection in plant cells. (A, B) REM photo of conventional (A) and newly developed glass pipette (B). (C) Fluorescent protein after injection into the nucleus of a plant cell. (D-F) Injection into chloroplasts (organelles in plant cells); Chlorophyll (D) and GFP fluorescence (E) and overlapping of the two (F).