Construction of marker-free transplastomic plants.

Because of its prokaryotic-type gene expression machinery, maternal inheritance and the opportunity to express proteins at a high level, the plastid genome (plastome or ptDNA) is an increasingly popular target for engineering. The ptDNA is present as up to 10,000 copies per cell, making selection for marker genes essential to obtain plants with uniformly transformed ptDNA. However, the marker gene is no longer desirable when homoplastomic plants are obtained. Marker-free transplastomic plants can now be obtained with four recently developed protocols: homology-based excision via directly repeated sequences, excision by phage site-specific recombinanses, transient cointegration of the marker gene, and the cotransformation-segregation approach. Marker excision technology will benefit applications in agriculture and in molecular farming.