Transformation Of Protoplasts

Protoplasts are plant cells from which the cell wall has been enzymatically or mechanically removed. As with animal cells, the contents of the protoplast cytoplasm are enclosed in a cell membrane, and the transformation of protoplasts can be achieved using many of the procedures routinely used to transfect cultured animal cells. Two procedures that are commonly used to introduce DNA into protoplasts include the uptake of naked DNA mediated by polyethylene glycol and a divalent cation (either Ca2+ or Mg2+) (50,51) and electroporation (52), although other agents such as lipofectin have also been used (53). Plant protoplasts can also be transformed using Agrobac-terium. Following transformation, protoplasts are placed on selective medium and allowed to regenerate new cell walls. The cells then proliferate

GFP (green fluorescent protein)

and form a callus from which embryos or shoots and roots can be regenerated with appropriate hormonal treatments (54).

In principle, protoplasts from any plant species can be transformed, but the technology is limited by the ability of protoplasts to regenerate into whole plants, which is not possible for all species. Although economical and potentially a very powerful procedure, direct transformation of protoplasts is disadvantageous because of the long culture times involved. Not only does this mean that the transformation process itself is time consuming, but cells cultured for extensive periods either fail to regenerate or frequently regenerate plants that show full or partial sterility and other phenotypic abnormalities (somaclonal variation). Protoplast preparation, maintenance, and transformation is a skilled technique, which, compared with Agrobacterium-mediated transformation and particle bombardment procedures, requires a greater investment in training. Advances in other transformation techniques are making protoplast transformation obsolete.

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