Direct Gene Uptake by Protoplasts - Protoplasts are cells without rigid cellulose walls. It has been shown that plant protoplasts treated with polyethylene glycol, commonly used to induce protoplast fusion, will take up DNA from their surrounding medium. More importantly, this DNA can then be stably integrated into the plant chromosomal DNA.

Using this technique, Potrykus and his colleagues (1985) at Frederich Meischer Institute induced cells of various plant species to incorporate an E.

coli gene directly; they used antibody selection techniques to prove that the gene is present and active in the plant cells.

At a certaiFl point, about a week after the DNAPEG treatment, kanamycin was added so that only cells with the kanamycin resistance trait actually completed development.

The evidence that the regenerated tobacco plants actually contained a gene for kanamycin resistance included the following:

(a) hybridization, on a Southern blot, of plant cell DNA to a radioactively labeled kanamycin resistance gene;

(b) presence of kanamycin phosphorylating activity, which inactivates the drug;

Thus it is likely that vectors will continue to be used in future experiments when possible.

Apart from polyethylene glycol, other agents used to protect the nucleic acid from nuclease digestion are poly (L-ornithine), poly (L-lysine), protamine sulphate, etc.

Direct gene uptake into protoplasts of some wheats, e.g; Triticum monococcum, has been successfully demonstrated. Direct gene transfer has also been effected in the grass Lolium multiflorum.

Protoplasts isolated from rice cells grown in suspension have been reported to be amenable to plasmid uptake in the presence of polyethylene glycol; the plasmid carries the gene to be transferred.

For example, chimeric marker genes have been transferred without the aid of vectors into protoplasts.

Various protocols are now available for introducing new genes into the protoplasts of a wide variety of plants, including the dicots, tobacco, petunia and carrot, and the monocots wheat and maize.

In all these cases the protoplasts are capable of developing into the undifferentiated type of tissue known as callus.

Whole plants that contain the transferred DNA can be regenerated from some of the callus tissue thus formed.

This works for the tobacco callus and that of other dicotyledonous plants but notably not for that of the cereals.

The foreign DNA often inserts at a single chromosomal location in the plant cells, although it less frequently integrates at multiple sites.

In most cases inheritance of the transferred DNA follows Mendelian rules.

How ever, the integrated DNA often undergoes various rearrangements, probably before it integrates in the plant genome, and this may make direct transfer of DNA protoplasts a less desirable means of introducing foreign DNA into plant cells than transfer by Agrobacterium.